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Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used for the delivery of nucleic acids (polynucleotides and oligonucleotides) to cells for therapeutic purposes that have been termed gene therapy or anti-sense therapy. One of the several methods of nucleic acid delivery to the cells is the use of DNA-polycations complexes. It was shown that cationic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine were effective intracellular delivery agents while small polycations like spermine were ineffective. (Felgner, P. L. (1990) Advanced Drug Delivery Rev. 5, 163-187; Boussif, O., Lezoualch, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) Proc. Natl. Acad. Sci. USA 92, 7297-7301) The mechanism by which polycations facilitate uptake of DNA is not completely understood but the following are some important principles:
1) Polycations provide attachment of DNA to the target cell surface: The polymer forms a cross-bridge between the polyanionic nucleic acids and the polyanionic surfaces of the cells. As a result the main mechanism of DNA translocation to the intracellular space might be non-specific adsorptive endocytosis which may be more effective then liquid endocytosis or receptor-mediated endocytosis. Furthermore, polycations are a very convenient linker for attaching specific receptors to DNA and as result, DNA-polycation complexes can be targeted to specific cell types.(Perales, J. C., Ferkol, T. Molas, M. and Hanson, W. (1994) Eur. J. Biochem. 226, 255-266; Cotten, M., Wagner, E. and Birnstiel, M. L. (1993) Methods in Enzymology 217, 618-644; Wagner, E., Curiel, D., and Cotten, M. (1994) Advanced Drug Delivery Rev. 114, 113-135).
2) Polycations protect DNA in complexes against nuclease degradation (Chiou, H. C., Tangco, M. V. Levine, S. M., Robertson, D., Kormis, K., Wu, C. H., and Wu, G. Y. (1994) Nucleic Acids Res. 22, 5439-5446). This is important for both extra- and intracellular preservation of DNA. The endocytic step in the intracellular uptake of DNA-polycation complexes is suggested by results in which DNA expression is only obtained by incorporating a mild hypertonic lysis step (either glycerol or DMSO) (Lopata, M. A., D. Clevland, W., and Sollner-Webb, B. (1984) Nucleic Acids Res. 12, 5707-5717; Golub, E. I., Kim, H. and Volsky, D. J. (1989) Nucleic Acid Res. 17, 4902). Gene expression is also enabled or increased by preventing endosome acidification with NH4CI or chloroquine (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res. 11, 1295-1300). Polyethylenimine which facilitates gene expression without additional treatments probably disrupts endosomal function itself (Boussif, O., Lezoualch, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) Proc. Natl. Acad. Sci. USA 92, 7297-7301). Disruption of endosomal function has also been accomplished by linking to the polycation endosomal-disruptive agents such as fusion peptides or adenoviruses (Zauner, W., Blaas, D., Kuechler, E., Wagner, E., (1995) J. Virology 69, 1085-1092; Wagner, E., Plank, C., Zatloukal, K., Cotten, M., and Birnstiel, M. L. (1992) Proc. Natl. Acad. Sci. 89, 7934-7938) (Fisher, K. J., and Wilson, J. M. (1994) Biochemical J. 299, 49-58).
3) Polycations generate DNA condensation: The volume which one DNA molecule occupies in complex with polycations is drastically lower than the volume of a free DNA molecule. The size of DNA/polymer complex is critical for gene delivery in vivo. In terms of intravenous injection, DNA needs to cross the endothelial barrier and reach the parenchymal cells of interest. The largest endothelia fenestrae (holes in the endothelial barrier) occurs in the liver and have an average diameter 100 nm. The fenestrae size in other organs is much lower. The size of the DNA complexes is also important for the cellular uptake process. After binding to the target cells the DNA-polycation complex should be taken up by endocytosis. Since the endocytic vesicles have a homogenous internal diameter of about 100 nm in hepatocytes of similar size in other cell types, the DNA complexes need to be smaller than 100 nm (Geuzze, H. J., Slot, J. W., Strous, G. J., Lodish, H. F., and Schwartz, A. L. (1982) J.Cell Biol. 92, 865-870).
Condensation of DNA
A significant number of multivalent cations with widely different molecular structures have been shown to induce the condensation of DNA. These include spermidine, spermine, Co(NH3)63+, protamine, histone H1, and polylysine.(Gosule, L. C. and Schellman, J. A. (1976) Nature 259, 333-335; Chattoraj, D. K., Gosule, L. C. and Schellman, J. A. (1978) J. Mol. Biol. 121, 327-337; Had, N. V., Downing, K. H. and Balhorn, R. (1993) Biochem. Biophys. Res. Commun. 193, 1347-1354; Hsiang, M. W and Cole, R. D. (1977) Proc. Natl. Acad. Sci. USA 74, 4852-4856; Haynes, M., Garret, R. A. and Gratzer, W. B. (1970) Biochemistry 9, 4410-4416; Widom, J. and Baldwin, R. L. (1980) J. Mol. Biol. 144, 431-453.). Quantitative analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized (Wilson, R. W. and Bloomfield, V. A. (1979) Biochemistry 18, 2192-2196). Depending upon the concentration of the DNA condensation leads to three main types of structures:
1) In extremely dilute solution (about 1 ug/ml or below), long DNA molecules can undergo a monomolecular collapse and form structures described as toroid.
2) In very dilute solution (about 10 ug/ml) microaggregates form with short or long molecules and remain in suspension. Toroids, rods and small aggregates can be seen in such solution.
3) In dilute solution (about 1 mg/ml, large aggregates are formed that sediment readily. (Sicorav, J.-L., Pelta, J., and Livolant, F (1994) Biophysical Journal 67, 1387-1392).
Toroids have been considered an attractive form for gene delivery because they have the lowest size. While the size of DNA toroids produced within single preparations has been shown to vary considerably, toroid size is unaffected by the length of DNA being condensed. DNA molecules from 400 bp to genomic length produce toroids similar in size (Bloomfield, V. A. (1991) Biopolymers 31, 1471-1481). Therefore one toroid can include from one to several DNA molecules. The kinetics of DNA collapse by polycations which resulted in toroids is very slow. For example DNA condensation by Co(NH3)6CI3 needs 2 hours at room temperature.(Arscott, P. G., Ma, C., and Bloomfield, V. A. (1995) Biopolymers 36, 345-364).
The mechanism of DNA condensation is not obvious. The electrostatic forces between unperturbed helices arise primarily from a counterion fluctuation mechanism requiring multivalent cations and plays the major role in DNA condensation.(Riemer, S. C. and Bloomfield, V. A. (1978) Biopolymers 17, 789-794; Marquet, R. and Houssier, C. (1991) J. Biomol. Struct. Dynam. 9, 159-167; Nilsson, L. G., Guldbrand, L. and Nordenskjold L. (1991) Mol. Phys. 72, 177-192). The hydration forces predominate over electrostatic forces when the DNA helices approach closer then a few water diameters (Leikin, S., Parsegian, V. A., Rau, D. C. and Rand, R. P. (1993) Ann. Rev. Phys. Chem. 44, 369-395). In case of DNA-polymeric polycation interactions, DNA condensation is a more complicated process than the case of low molecular weight polycations. Different polycationic proteins can generate toroid and rod formation with different size DNA at a ratio of positive to negative charge of 0.4 (Garciaramirez, M., and Subirana, J. A. (1994) Biopolymers 34, 285-292). It was shown by fluorescence microscopy that T4 DNA complexed with polyarginine or histone can forms two types of structures; an elongated structure with a long axis length of about 350 nm (like free DNA) and dense spherical particles. (Minagawa, K., Matsuzawa, Y., Yshikawa, K., Matsumoto, M., and Doi, M. (1991) FEBS Lett. 295, 60-67). Both forms exist simultaneously in the same solution. The reason for the co-existence of the two forms can be explained as an uneven distribution of the polycation chains among the DNA molecules. The uneven distribution generates two thermodynamically favorable conformations. Kabanov, A. V., and Kabanov, V. A. (1995) Bioconjugate Chem. 6, 7-20).
It was also shown that the electrophoretic mobility of DNA-polycation complexes can change from negative to positive in excess of polycation. It is likely that large polycations don""t completely align along DNA but form polymer loops which interact with other DNA molecules. The rapid aggregation and strong intermolecular forces between different DNA molecules may prevent the slow adjustment between helices needed to form tightly packed, orderly particles. This specification describes a new approach, that we have termed Polynucleotide Template Polymerization, for overcoming this problem of nonspecific aggregation and large DNA-polycation complex formation that occurs when polycation/DNA complexes are formed in DNA concentrations that are of practical value for polynucleotide transfer into cells and for gene or antisense therapy.
A process for drug delivery is described in which polymerization and chemical reaction processes are induced in the presence of the drug in order to deliver the drug or biologically active compound. Drug delivery encompasses the delivery of a biologically active compound to a cell. By xe2x80x9cdeliveringxe2x80x9d we mean that the drug becomes associated with the cell. The drug can be on the membrane of the cell or inside the cytoplasm, nucleus, or other organelle of the cell. The process of delivering a polynucleotide to a cell has also been commonly termed xe2x80x9ctransfectionxe2x80x9d or the process of xe2x80x9ctransfectingxe2x80x9d and also it has been termed xe2x80x9ctransformationxe2x80x9d. A biologically active compound is a compound having the potential to react with biological components. Pharmaceuticals, proteins, peptides and nucleic acids are examples of biologically active compounds. The template polymer can be a polyanion such as a nucleic acid. The polynucleotide could be used to produce a change in a cell that can be therapeutic. The delivery of polynucleotides or genetic material for therapeutic purposes is commonly called xe2x80x9cgene therapyxe2x80x9d.
A new method is described for forming condensed nucleic acid by having a chemical reaction take place in the presence of the nucleic acid. A process is also described of forming in the presence of the nucleic acid a polymer that has affinity to nucleic acid. Moreover, a process is described of forming an interpolyelectrolyte complex containing nucleic acids by having a chemical reaction take place in the presence of the nucleic acid. In addition, the nucleic acid-binding polymer can form as a result of template polymerization. This obviously excludes the formation of polymers such as proteins or nucleic acids or other derivatives that bind nucleic acid by Watson-Crick binding.
Previously, the occurrence of chemical reactions or the process of polymerization in the presence of the nucleic acid has been assiduously avoided when delivering nucleic acid. Perhaps, this arose out of concerns that the processes of chemical reactions or polymerization would chemically modify the nucleic acid and thereby render it not biologically active. Surprisingly, we show that we can perform polymerizations in the presence of nucleic acids without chemically modifying the nucleic acid and that the nucleic acid is still functional. For example, a plasmid construct containing a promoter and the reporter gene luciferase can still express as much luciferase as native plasmid after transfection into cells.
The process of forming a polymer in the presence of nucleic acid has several advantages. As FIG. 1 illustrates, aggregation and precipitation of the nucleic acid can be avoided by having the polymerization take place in the presence of the nucleic acid. This newly described process enabled us to form supramolecular complexes of nucleic acid and polymer rapidly, consistently, and at very high concentrations of polynucleic acid. In fact, high concentration of the template nucleic acid favors this process. In contrast, the previously described process of mixing a nucleic acid and an already-formed polycation (such as polylysine) has to be done at very dilute concentrations. In addition, the previously-described procedure requires that the mixing, salt and ionicity conditions must be carefully controlled as well. This explains why the use of polylysine-DNA complexes are not widely used for the transfer of DNA into cells and is only done in a few laboratories.
The other advantage that flows from the newly described process of having polymerization take place in the presence of nucleic acid is that polymers could form that would not be able to become associated with nucleic acids if the polymer was formed first. For example, the polymerization process could result in a hydrophobic polymer that is not soluble in aqueous solutions unless it is associated with nucleic acid. A hydrophobic moiety comprises a C6-C24 alkane, C6-C24 alkene, sterol, steroid, lipid, or hydrophobic hormone. Furthermore, the process of having the polymerization taking place in organic solvents and heterophase systems enables more types and more defined types of vesicles to be formed.
This process will enable supramolecular complexes to be more easily assembled. It will also enable novel and more defined complexes to be made. Yet another advantage that flows from this invention is that nucleic acid/polymer complexes will be smaller. The size of DNA/polymer complex is critical for gene delivery especially in vivo.
These processes can be used for transferring nucleic acids into cells or an organism such as for drug delivery. They may also be used for analytical methods or the construction of new materials. They may also be used for preparative methods such as in the purification of nucleic acids. They are also useful for many types of recombinant DNA technology. For example, they may be used to generate sequence binding molecules and protect specific sequences from nuclease digestion. Protection of specific regions of DNA is useful in many applications for recombinant DNA technology.
A preferred embodiment provides a method of making a compound for delivery to a cell, comprising: forming a polymer in the presence of a biologically active drug.
Another preferred embodiment provides a method of making a compound for delivery to a cell, comprising: cross-linking a polymer in the presence of a polyion, thereby forming a complex of polymer and polyion; and, delivering the complex to the cell.
Another preferred embodiment provides a method of making a compound for delivery to a cell, comprising: modifying a molecule in the presence of the polyion thereby providing a deliverable polyion.
Yet, another preferred embodiment provides a method of making a compound for delivery to a cell, comprising: mixing a polyion with a first polymer and a second polymer thereby forming a deliverable complex.
Further objects, features, and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.